Compounds and compositions for the treatment of muscular disorders and bone disorders

ABSTRACT

The present invention relates to compounds and compositions and their applications as pharmaceuticals for treating, preventing, or reversing injury to skeletal or cardiac muscles, for treating or preventing diseases relating to the structure and function of skeletal or cardiac muscle, and for inducing regeneration or restructuring of skeletal or cardiac muscle as a means of treating diseases relating to abnormalities in skeletal or cardiac muscle structure and function in a human or animal subject. Further aspects relate to the same or similar applications for bone and bone diseases.

This application is a continuation of U.S. patent application Ser. No.16/742,608, filed Jan. 14, 2020, which is a continuation of U.S. patentapplication Ser. No. 15/900,533, filed Feb. 20, 2018, which is acontinuation in part of U.S. patent application Ser. No. 14/387,117,which adopts an international filing date of Mar. 22, 2013, now U.S.Pat. No. 9,901,564 which is the 371 national stage filing ofPCT/US2013/033555, filed Mar. 22, 2013, which claims the benefit of U.S.Provisional Patent Application No. 61/614,721, filed Mar. 23, 2012, thedisclosures of which are hereby incorporated by reference as if writtenherein in their entireties.

Disclosed herein are compounds and compositions and their application aspharmaceuticals for treating, preventing, or reversing injury toskeletal or cardiac muscles, for treating or preventing diseasesrelating to the structure and function of skeletal or cardiac muscle,and for inducing regeneration or restructuring of skeletal or cardiacmuscle as a means of treating diseases relating to abnormalities inskeletal or cardiac muscle structure and function in a human or animalsubject. Also disclosed herein are methods for diagnosing injury toskeletal or cardiac muscle and for diagnosing the success or failure oftherapeutics designed to treat, prevent, or reverse injury to skeletalmuscle or cardiac muscle.

Strength and endurance of skeletal muscle is essential for gripping,carrying, walking, running, carrying or enabling numerous functions ofeveryday life. Strength and endurance of cardiac muscle is essential forthe optimum delivery of oxygen and nutrients to all tissues containingblood vessels and for the carrying away of waste products of cellmetabolism. Injury to skeletal or cardiac muscle or diseases relating toabnormal structure or function of skeletal or cardiac muscle can makenormal activities of everyday life difficult or impossible.

Further, injury to, or weakness of, skeletal muscle generally results ina loss of bone density in the bones to which that muscle is attached. Inthe case of generalized muscle weakness, reduction in bone density canbe generalized, one of the causes of the bone disease known asosteoporosis.

Injury to skeletal or cardiac muscle can occur as a result of geneticmutations in proteins critical to the structure and function of skeletalmuscle or cardiac muscle, inadequate or interrupted blood flow,inactivity due to joint injury or inflammation, as is seen witharthritis, excessive exposure to oxidation injury as a result ofdefective cell metabolism or inadequate blood flow, exposure to toxicorganic or inorganic substances such as elevated glucose, heavy metals,or inflammatory products, trauma due to injury or excessive activity, orexposure to certain medications such as statins, corticosteroids, orchemotherapy, among other causes. Examples of inflammatory diseasesassociated with muscle disorders include polymyositis, polymyalgiarheumatic a, and systemic lupus erythematosus.

Injury to skeletal muscle and consequent weakness or atrophy can occuras a result of injury or disorders of the neurons subserving musclefunction. Appropriate innervation is essential to skeletal muscle healthand function. Neurodegenerative diseases amenable to treatment withagents stimulating muscle strength and neuromuscular health includeamyotrophic lateral sclerosis (ALS), Parkinson's disease, Huntington'sdisease, spinal cord injury or abnormality, and peripheral and centralneuropathies.

Currently therapies emphasize prevention, such as use of stents toimprove blood flow through areas of vascular narrowing. There aregeneral supportive interventions to help the muscle repair itself, suchas the nutritional provision of muscle protein precursors such as aminoacids or creatine. Current therapies may address the underlying disorderassociated with cardiac or skeletal muscle dysfunction without directlytreating the muscle cells themselves. The only accepted therapy directedat the muscles themselves is exercise. It has been demonstrated thatregular, moderate activation of muscle cells can improve the structureand function of cardiac and skeletal muscle cells. However, this isoften inadequate in restoring muscle cell health or function.

Complicating the potential therapies is the fact that neither skeletalmuscle nor cardiac muscle cells are capable of sufficient proliferationin order to replace muscle cells previously damaged or destroyed. Theremay be some limited capacity of stem cells to proliferate but this isnot generally sufficient to regenerate functionally significantreplacement muscle. Skeletal muscle is known to contain primitivesatellite cells, which can activate, enlarge, and differentiate intoskeletal muscle tissue. The role of satellite cells in replacing cardiaccells is currently not well understood. Repair of muscles is enhanced bymuscle cellular expression of folistatin, which allows for activationand differentiation of muscle precursor cells into mature,differentiated skeletal muscle cells. Repair of muscle cells orgeneration of new, differentiated muscle cells is inhibited by theexpression of a negative regulatory factor known as myostatin.

Disclosed herein are methods for prophylactic and/or therapeutictreatment of skeletal or cardiac muscle dysfunction, injury, or diseasesin a patient by administering epicatechin, an epicatechin derivative, apharmaceutically acceptable salt or prodrug thereof, or combinationsthereof. The methods and compositions described herein can assist inprevention of impaired skeletal and cardiac muscle function, recovery ofskeletal or cardiac muscle health or function, or functionallysignificant regeneration of skeletal or cardiac muscle cells orfunction.

In certain embodiments, the present invention comprises administering acompound or composition disclosed herein in an amount effective tostimulate function, recovery, or regeneration of skeletal or cardiacmuscle cells. Stimulation of muscle cell function, recovery, orregeneration may comprise increased expression of one or more ofproteins having contractile, regulatory, transcriptional, or attachmentfunctions. Stimulation of muscle cell function, recovery, orregeneration may comprise increased mitochondrial number and function.In certain embodiments, the compound or composition comprises atherapeutically effective amount of epicatechin, either (+) or (−)enantiomers or a combination of both, an epicatechin derivative, or apharmaceutically acceptable salt or prodrug thereof.

In further embodiments, the present invention provides methods andcompositions for preventing or treating adverse events or diseasesassociated with impaired skeletal muscle or cardiac muscle cell numberor function. The methods comprise administering to a subject in needthereof one or more compounds or compositions disclosed herein. Infurther embodiments the method reduces symptoms of impaired skeletal orcardiac muscle cell number or function. In certain embodiments, themethod comprises administering, or the composition comprises, atherapeutically effective amount of epicatechin, either (+) or (−)enantiomers or a combination of both, an epicatechin derivative, or apharmaceutically acceptable salt or prodrug thereof.

In certain embodiments, disclosed herein are methods and compositionsfor the treatment of diseases associated with loss of number, function,or correct, optimally efficient internal organization of skeletal musclecells or cardiac muscle cells. In certain embodiments, the methodcomprises administering, or the composition comprises, a therapeuticallyeffective amount of epicatechin, either (+) or (−) enantiomers or acombination of both, an epicatechin derivative, or a pharmaceuticallyacceptable salt or prodrug thereof.

In further embodiments, disclosed herein are methods and compositionsfor the treatment of impaired skeletal or cardiac muscle function due toaging, obesity, disuse or inactivity, exposure to potentially toxicnutritional agents such as fructose, or exposure to inadequate nutritionsuch as starvation or malnutrition. In certain embodiments, the methodcomprises administering, or the composition comprises, a therapeuticallyeffective amount of epicatechin, either (+) or (−) enantiomers or acombination of both, an epicatechin derivative, or a pharmaceuticallyacceptable salt or prodrug thereof.

In further embodiments, disclosed herein are methods and compositionsfor the treatment of muscle-related side effects of athletic training orcompetition including soreness, cramping, weakness, pain, or injury. Incertain embodiments, the method comprises administering, or thecomposition comprises, a therapeutically effective amount ofepicatechin, either (+) or (−) enantiomers or a combination of both, anepicatechin derivative, or a pharmaceutically acceptable salt or prodrugthereof.

In further embodiments, disclosed herein are methods and compositionsfor the treatment of skeletal or cardiac muscle diseases associated withischemia, or impaired or inadequate blood flow. Examples of such statesinclude, but are not limited to, atherosclerosis, trauma, diabetes,vascular stenosis, peripheral arterial disease, vasculopathy, andvasculitis. In certain embodiments, the method comprises administering,or the composition comprises, a therapeutically effective amount ofepicatechin, either (+) or (−) enantiomers or a combination of both, anepicatechin derivative, or a pharmaceutically acceptable salt or prodrugthereof.

In further embodiments, disclosed herein are methods and compositionsfor the treatment of diseases associated with genetic disorders thatdirectly or indirectly affect the number, structure, or function ofcardiac muscle cells or skeletal muscle cells. Examples of such statesinclude, but are not limited to, the set of diseases broadly classifiedas muscular dystrophies and Friedreich's ataxia. In certain embodiments,the method comprises administering, or the composition comprises, atherapeutically effective amount of epicatechin, either (+) or (−)enantiomers or a combination of both, an epicatechin derivative, or apharmaceutically acceptable salt or prodrug thereof.

In further embodiments, disclosed herein are methods and compositionsfor the therapeutic treatment of diseases associated with impairedneurological control of muscular activity resulting in consequentabnormalities in structure and function of skeletal muscles due toinactivity, aberrant contractility, or contracted states. These include,but are not limited to, states associated with absent, diminished, orabnormal neurological activity including peripheral denervationsyndromes, trauma, amyotrophic lateral sclerosis, meningitis, andstructural abnormalities of the spine, whether congenital or acquired.In certain embodiments, the method comprises administering, or thecomposition comprises, a therapeutically effective amount ofepicatechin, either (+) or (−) enantiomers or a combination of both, anepicatechin derivative, or a pharmaceutically acceptable salt or prodrugthereof.

In certain embodiments, disclosed herein are methods and compositionsfor the treatment of diseases associated with loss of number, loss offunction, or loss of correct, optimally efficient internal organizationof skeletal muscle cells or cardiac muscle cells. Such diseases mayeventuate in a state of functionally significant muscle wasting, which,in its most pronounced form, is termed sarcopenia. Sarcopenia may besecondary to a variety of disorders, including aging, diabetes or otherabnormal metabolic conditions, infection, inflammation, autoimmunedisease, cardiac dysfunction, or severe disuse syndromes or inactivityassociated with arthritis. Examples of such diseases include, but arenot limited to, congestive heart failure, aging, myocarditis, myositis,polymyalgia rheumatic, polymyositis, HIV, cancer and/or the side effectsof chemotherapy targeting the cancer, malnutrition, aging, inborn errorsof metabolism, trauma, and stroke or other types of neurologicalimpairment. In certain embodiments, the method comprises administering,or the composition comprises, a therapeutically effective amount ofepicatechin, either (+) or (−) enantiomers or a combination of both, anepicatechin derivative, or a pharmaceutically acceptable salt or prodrugthereof.

In certain embodiments, disclosed herein are methods and compositionsfor use in combination with exercise or programmatic sequences orintensities of exercise to optimize methods for the prophylactic ortherapeutic treatment of diseases or disorders associated with loss ofnumber, loss of function, or loss of correct, optimally efficientinternal organization of skeletal muscle or cardiac muscle cells. Incertain embodiments, the method comprises administering, or thecomposition comprises, a therapeutically effective amount ofepicatechin, either (+) or (−) enantiomers or a combination of both, anepicatechin derivative, or a pharmaceutically acceptable salt or prodrugthereof.

In certain embodiments, disclosed herein are methods and compositionsfor use to enhance sports performance and endurance, to build muscleshape and strength, and to facilitate recovery from the muscle relatedside effects of training or competition, such as soreness, weakness,cramping, pain, or injury. In certain embodiments, the method comprisesadministering, or the composition comprises, a therapeutically effectiveamount of epicatechin, either (+) or (−) enantiomers or a combination ofboth, an epicatechin derivative, or a pharmaceutically acceptable saltor prodrug thereof.

In certain embodiments, disclosed herein are methods and compositionsfor use to prevent, ameliorate, or reverse muscle injury, weakness, orpain associated with the administration of certain medicines, including,but not limited to, corticosteroids such as prednisone, methylprednisone, or halogenated derivatives thereof, chemotherapeutics suchas doxorubicin or methotrexate, and inhibitors of HMG co-reductase,known as statins, that are frequently associated with muscle disordersor myopathy, including: Advicor′″ (niacin extended-release/lovastatin),Altoprev′″ (lovastatin extended-release), Caduet′″ (amlodipine andatorvastatin), Crestor′″ (rosuvastatin), Juvisync′″(sitagliptin/simvastatin), Lescol′″ (fluvastatin), Lescol XL(fluvastatin extended-release), Lipitor″ (atorvastatin), Compactin(mevastatin), Livalo® (pitavastatin), Mevacor′″ (lovastatin),Pravachol′″ (pravastatin), Simcor′″ (niacinextended-release/simvastatin), Vytorin′″ (ezetimibe/simvastatin), andZocor′″ (simvastatin). In certain embodiments, the method comprisesadministering, or the composition comprises, a therapeutically effectiveamount of epicatechin, either (+) or (−) enantiomers or a combination ofboth, an epicatechin derivative, or a pharmaceutically acceptable saltor prodrug thereof.

In certain embodiments, disclosed herein are methods and compositionsfor use to prevent, ameliorate, or reverse muscle injury associated withmedicines that damage mitochondria and/or cause myopathy as a secondaryconsequence.

In certain embodiments, a subject is selected for treatment with acompound or composition disclosed herein based on the occurrence of oneor more physiological manifestations of skeletal or cardiac muscleinjury or dysfunction in the subject. Such manifestations includeelevations in biomarkers known to be related to injury of the heart orskeletal muscle. Examples of such biomarkers include, but are notlimited to, elevated plasma levels of cardiac or skeletal muscle enzymesor proteins, such as myoglobin, troponin, or creatine phosphokinase,lactic acidosis, and elevated serum creatinine.

In certain embodiment, a compound or composition as disclosed herein isadministered in an amount which stimulates increased number or functionof skeletal muscle cells or contractile muscle cells. Such stimulationof muscle cells may comprise stimulation of one or more aspects ofmuscle cell function, including cell division, muscle cell regeneration,activation of muscle satellite cells and their differentiation intoadult muscle cells, recovery from injury, increased number or functionof mitochondria or processes serving mitochondrial function, increasedexpression of proteins contributing to contractility, regulation ofbiochemical or translational processes, mitoses, or transduction ofmechanical energy via dystrophin or other attachment processes. Themethods and compositions described herein can assist in prevention ofthe consequences of muscle injury or dysfunction which have not yetoccurred, as well as provide for the active therapy of muscle injury,dysfunction, or diseases which have already occurred.

In certain embodiments, disclosed herein are methods to utilize themuscle proteins whose expression is stimulated by administration ofcompounds or compositions disclosed herein as diagnostic biomarkers bywhich to determine the time and degree of muscle response to thetherapeutic methods and compositions disclosed herein. Such biomarkersmay be determined by measuring in tissue, plasma, blood, or urine theproteins themselves or the DNA or RNA nucleotides that encode for theproteins. In one embodiment, a decrease in the body of useful muscleproteins, such as dystrophin, or the presence of inhibitory proteins,such as thromobospondin, may be used to diagnose the severity of theabnormality of cardiac muscle structure or function or the probabilityof response to the therapeutic methods and compositions describedherein. In another embodiment, changes in the levels of such biomarkersmay be used to gauge the success or failure of certain therapeuticmodalities, including those disclosed herein, in order to optimize thedose and to decide whether to maintain or change therapeutic methods andcompositions.

In another embodiment, an increase in the plasma concentration offollistatin, or a decrease in myostatin, or an increase in the ratio ofplasma follistatin to plasma myostatin, may be used as a diagnosticmethod to diagnose the degree of severity of a muscle disorder or theextent of response to therapy.

In certain embodiments, the methods disclosed herein comprise theadministration to cells at least 0.1) 1M epicatechin or an epicatechinderivative, at least 0.25)1M epicatechin or an epicatechin derivative,at least 0.5) 1M epicatechin or an epicatechin derivative, and atleast 1) 1M epicatechin or an epicatechin derivative.

In further embodiments, the methods disclosed herein comprise theadministration of compounds of the disclosure in a total daily dose ofabout 0.1 mg/kg/dose to about 100 mg/kg/dose, alternately from about 0.3mg/kg/dose to about 30 mg/kg/dose. In another embodiment the dose rangeis from about 0.5 to about 10 mg/kg/day. Alternately about 0.5 to about1 mg/kg/day is administered. Generally between about 25 mg and about 1gram per day can be administered; alternately between about 25 mg andabout 200 mg can be administered. The dose may be administered in asmany divided doses as is convenient.

In further embodiments, the methods disclosed herein comprise theadministration of epicatechin, an epicatechin derivative, or a mixturethereof in a range of about 1 to about 1000 mg per kg body weight, about1 to about 50 mg per kg body weight, or about 10 to about 100 mg per kgbody weight of said subject.

In further embodiments, the desired concentration is maintained for atleast 30 minutes, 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 48hours, 72 hours, or more. In yet further embodiments, the desiredconcentration is achieved at least once during each 12-hour period overat least 24 hours, 48 hours, 72 hours, 1 week, one month, or more; or atleast once during each 24-hour period over at least 48 hours, 72 hours,1 week, one month, or more. In order to maintain a desired concentrationfor a desired time, multiple doses of one or more compounds may beemployed. The dosing interval may be determined based on the clearancehalf-life for each compound of interest from the body.

In certain embodiments, the epicatechin or epicatechin derivativeadministered in a method disclosed herein is at least 90% pure relativeto other compounds selected from the group consisting of epicatechin, anepicatechin derivative, catechin, or a catechin derivative. For example,if the compound is epicatechin, it contains no more than 10%contamination with epicatechin derivatives, catechin, and catechinderivatives. In further embodiments the selected epicatechin orepicatechin derivative is at least 95% pure relative to other compoundsselected from the group consisting of epicatechin, an epicatechinderivative, catechin, or a catechin derivative. It is noted that thisdoes not exclude combination with an additional therapeutic agent insubstantial concentration.

In further embodiments, said epicatechin is (−)-epicatechin.

In further embodiments, said epicatechin is (+)-epicatechin.

In further embodiments, said epicatechin is a racemic mixture of(−)-epicatechin and (+)-epicatechin.

Also disclosed herein is a novel class of compounds that are the onlyknown agents that, when administered, induce the production ofcirculating follistatin in the body of animals and humans. Follistatinand its closely homologous (80% sequence homology) family offollistatin-like proteins are produced by numerous cell types in thebody, including muscle cells and bone cells. Follistatin is known toinduce muscle regeneration in various disease states. Follistatin-likeprotein 3 has recently been shown to stimulate bone regeneration andincrease mechanical bone strength in exercise. See J Nam et. Al.Follistatin-like 3 is a mediator of exercise-driven bone formation andstrengthening. Bone 2015 78:62-70 doi:10.1016/j.bone.2015.04.038.Exercise, which strengthens both muscle and bone, induces increasedplasma levels of follistatin and its related, follistatin-like proteins.See Hansen J et al. Exercise induces a marked increase in plasmafollistatin: evidence that follistatin is a contraction-inducedhepatokine. 2011 Endocrinology 152:164-171 Pub Med 21068158. Animalslacking follistatin display weak skeletal bone formation and profoundmuscle weakness. See Matzuk, M M et al. Multiple defects and perinataldeath in mice deficient in follistatin. Nature 1995 374:360-3. PubMed7885475. Production of extracellular matrix and its mineralization arethe essential components of new bone formation by osteoblasts.Follistatin stimulates both activities in cultures of human osteoblasts.See Eijken et al, The activin A-follistatin system: potent regulator ofhuman extracellular matrix mineralization Faseb J 2007 21:2949-60.During the healing of bone fractures, the expression of follistatin andits receptors are strongly increased in the periosteum near the ends ofthe bone fractures, indicating that follistatin is contributing to theformation and remodeling of bone during fracture healing. See Nagame Tet al Immunochemical detection of activin A, follistatin, and activinreceptors during fracture healing in the rat J Orthop Res 199816:314-21. Thus any agent that stimulates follistatin production shouldbe therapeutic in the context of the numerous diseases, conditions, drugside effects, and genetic defects that contribute to the development ofosteoporosis and its attendant increased risk of bone fractures.

Some embodiments relate to a method to induce increased cellular ormuscular or bodily production of follistatin and follistatin-likeproteins in order to reverse or ameliorate injury to, or weakness of, orloss of, bone, or to prevent fractures, in a subject in need thereof,comprising administering to a subject a therapeutically effective doseof (+)-epicatechin and/or (−)-epicatechin or an epicatechin derivativeof either (+)-epicatechin or (−)-epicatechin. In further embodiments,the (+)-epicatechin and/or (−)-epicatechin or an epicatechin derivativeis administered orally or intravenously or intramuscularly, at 5 mg to 2grams per day, in a single dose or in divided doses. In otherembodiments, the (+)-epicatechin and/or the (−)-epicatechin or anepicatechin derivative is administered at a dose between 0.1 mg/kg ofbodyweight per day to 10 mg/kg of bodyweight per day, orally orintravenously or intramuscularly, in a single dose or in divided doses.In still other embodiments, the method induces new bone formation oradditional bone formation or stronger bone formation or regeneration ofbone in order to prevent bone fractures.

Additional embodiments disclosed herein relate to a method to induce theincreased cellular or muscular or bodily production of follistatin andfollistatin-like proteins in order to reverse or ameliorate weakness ofbone, thus preventing bone fractures, caused by administration ofcompounds known to induce weakness of or damage to bone, impairment ofbone generation, or impairment of bone growth, including but not limitedto corticosteroids such as prednisone, or deflazacort, anticonvulsantssuch as phenytoin and phenobarbital, chemotherapeutics such as aromataseinhibitors, and progestins. Further method aspects relate to a to inducethe increased cellular or muscular or bodily production of follistatinor follistatin-like proteins in order to reverse or ameliorate weaknessof bone strength, thus preventing bone fractures, associated withgenetic predisposition, aging, inactive lifestyle, or low estrogenstates such as menopause or post oophorectomy; a method to induce theincreased cellular or muscular or bodily production of follistatin orfollistatin-like proteins in order to reverse or ameliorate weakness ofbone caused by medical conditions known to be associated with weaknessof, or damage to, bone, impairment of bone generation, or impairment ofbone growth, such as celiac disease, kidney or liver disease, andinflammatory diseases such as systemic lupus erythematosus andrheumatoid arthritis; a method to induce the increased cellular ormuscular or bodily production of follistatin or follistatin-likeproteins in order to reverse or ameliorate weakness of bone inconjunction with the administration of other agents used to treatosteoporosis including calcium, Vitamin D, and calcitonin, in order toprevent bone fractures; a method to induce increased cellular ormuscular or bodily production of follistatin or follistatin-likeproteins as a therapeutic to accelerate the healing of bone fractures orto increase the degree of recovery from a bone fracture, such as thoseexperienced in accidents, athletics, or combat; and a method to induceincreased cellular or muscular or bodily production of follistatin orfollistatin-like proteins in order to prevent systemic loss of bonedensity, and thus prevent subsequent bone fractures, during the recoveryperiod after orthopedic surgery or after the onset of a disease orcondition necessitating long periods of bed rest or physical inactivity,which are known to result in decreased bone density and muscle weakness.

In any of the aforementioned method embodiments, it is contemplated thatthe (+)-epicatechin and/or (−)-epicatechin or an epicatechin derivativemay optionally be administered orally or intravenously orintramuscularly; at 5 mg to 2 grams per day; and/or in a single dose orin divided doses.

DESCRIPTION OF DRAWINGS

FIG. 1 shows pictures of mice in the various treatment groups.

FIG. 2 shows additional pictures of mice in the various treatmentgroups.

FIG. 3 depicts a chart of bone length in four different treatmentgroups.

DETAILED DESCRIPTION

Accordingly, provided herein is a method of treating, preventing, orreversing injury to skeletal or cardiac muscles, comprising theadministration of a therapeutically effective amount of epicatechin,either (+) or (−) enantiomers or a combination of both, an epicatechinderivative, a pharmaceutically acceptable salt or prodrug thereof, orcombinations thereof to a patient in need thereof. Also provided is acomposition for treating, preventing, or reversing injury to skeletal orcardiac muscles, comprising the administration of a therapeuticallyeffective amount of epicatechin, either (+) or (−) enantiomers or acombination of both, an epicatechin derivative, a pharmaceuticallyacceptable salt or prodrug thereof, or combinations thereof to a patientin need thereof.

Also provided is a method of treating a disease relating to an impairedskeletal or cardiac muscle structure or function of skeletal or cardiacmuscle, comprising the administration of a therapeutically effectiveamount of epicatechin, either (+) or (−) enantiomers or a combination ofboth an epicatechin derivative, a pharmaceutically acceptable salt orprodrug thereof, or combinations thereof to a patient in need thereof.Also provided is a composition for treating, preventing, or reversinginjury to skeletal or cardiac muscles, comprising a therapeuticallyeffective amount of epicatechin, either (+) or (−) enantiomers or acombination of both, an epicatechin derivative, a pharmaceuticallyacceptable salt or prodrug thereof, or combinations thereof to a patientin need thereof.

In certain embodiments, said impairment is due to aging, obesity, disuseor inactivity, exposure to potentially toxic nutritional agents such asfructose, or exposure to inadequate nutrition such as starvation ormalnutrition.

Also provided herein is a method of inducing regeneration orrestructuring of skeletal or cardiac muscle, comprising theadministration of a therapeutically effective amount of epicatechin,either (+) or (−) enantiomers or a combination of both an epicatechinderivative, a pharmaceutically acceptable salt or prodrug thereof, orcombinations thereof to a patient in need thereof. Also provided is acomposition for inducing regeneration or restructuring of skeletal orcardiac muscle, comprising a therapeutically effective amount ofepicatechin, either (+) or (−) enantiomers or a combination of both anepicatechin derivative, a pharmaceutically acceptable salt or prodrugthereof, or combinations thereof to a patient in need thereof.

Also provided herein is a method of diagnosing injury to skeletal orcardiac muscle and for diagnosing the success or failure of therapeuticsdesigned to treat, prevent, or reverse injury to skeletal muscle orcardiac muscle, comprising:

-   -   a. observing one or more physiological manifestations of        skeletal or cardiac muscle injury or dysfunction in the subject;    -   b. administering a therapeutically effective amount of        epicatechin, either (+) or (−) enantiomers or a combination of        both, an epicatechin derivative, a pharmaceutically acceptable        salt or prodrug thereof, or combinations thereof to a patient in        need thereof; and    -   c. observing a change or lack thereof in said physiological        manifestations of skeletal or cardiac muscle injury or        dysfunction.

Also provided herein is a method of improving muscle cell function,recovery, or regeneration, comprising the administration of atherapeutically effective amount of epicatechin, either (+) or (−)enantiomers or a combination of both, an epicatechin derivative, apharmaceutically acceptable salt or prodrug thereof, or combinationsthereof to a patient in need thereof. Also provided is a composition forimproving muscle cell function, recovery, or regeneration, comprising atherapeutically effective amount of epicatechin, either (+) or (−)enantiomers or a combination of both, an epicatechin derivative, apharmaceutically acceptable salt or prodrug thereof, or combinationsthereof to a patient in need thereof.

In certain embodiments, improving of muscle cell function, recovery, orregeneration comprises increased mitochondrial number and function.

Also provided herein is a method of treating muscle-related side effectsof athletic training or competition including soreness, cramping,weakness, pain, or injury, comprising the administration of atherapeutically effective amount of epicatechin, either (+) or (−)enantiomers or a combination of both, an epicatechin derivative, apharmaceutically acceptable salt or prodrug thereof, or combinationsthereof to a patient in need thereof. Also provided is a composition fortreating muscle-related side effects of athletic training or competitionincluding soreness, cramping, weakness, pain, or injury, comprising atherapeutically effective amount of epicatechin, either (+) or (−)enantiomers or a combination of both, an epicatechin derivative, apharmaceutically acceptable salt or prodrug thereof, or combinationsthereof to a patient in need thereof.

Also provided herein is a method of treating skeletal or cardiac musclediseases associated with ischemia or impaired or inadequate blood flow,comprising the administration of a therapeutically effective amount ofepicatechin, either (+) or (−) enantiomers or a combination of both, anepicatechin derivative, a pharmaceutically acceptable salt or prodrugthereof, or combinations thereof to a patient in need thereof. Alsoprovided is a composition for treating skeletal or cardiac musclediseases associated with ischemia or impaired or inadequate blood flow,comprising a therapeutically effective amount of epicatechin, either (+)or (−) enantiomers or a combination of both, an epicatechin derivative,a pharmaceutically acceptable salt or prodrug thereof, or combinationsthereof to a patient in need thereof.

In certain embodiments, said diseases are selected from the groupconsisting of atherosclerosis, trauma, diabetes, vascular stenosis,peripheral arterial disease, vasculopathy, and vasculitis.

Also provided herein is a method of treating a disease associated withgenetic disorders that directly or indirectly affect the number,structure, or function of cardiac muscle cells or skeletal muscle cells,comprising the administration of a therapeutically effective amount ofepicatechin, either (+) or (−) enantiomers or a combination of both, anepicatechin derivative, a pharmaceutically acceptable salt or prodrugthereof, or combinations thereof to a patient in need thereof. Alsoprovided is a composition for treating diseases associated with geneticdisorders that directly or indirectly affect the number, structure, orfunction of cardiac muscle cells or skeletal muscle cells, comprisingtherapeutically effective amount of epicatechin, either (+) or (−)enantiomers or a combination of both, an epicatechin derivative, apharmaceutically acceptable salt or prodrug thereof, or combinationsthereof to a patient in need thereof.

In certain embodiments, said diseases are selected from the groupconsisting of muscular dystrophies and Friedreich's ataxia.

Also provided herein is a method of treating diseases associated withimpaired neurological control of muscular activity resulting inconsequent abnormalities in structure and function of skeletal musclesdue to inactivity, aberrant contractility, or contracted states,comprising the administration of a therapeutically effective amount ofepicatechin, either (+) or (−) enantiomers or a combination of both, anepicatechin derivative, a pharmaceutically acceptable salt or prodrugthereof, or combinations thereof to a patient in need thereof. Alsoprovided is a composition for treating diseases associated with impairedneurological control of muscular activity resulting in consequentabnormalities in structure and function of skeletal muscles due toinactivity, aberrant contractility, or contracted states, comprising atherapeutically effective amount of epicatechin, either (+) or (−)enantiomers or a combination of both, an epicatechin derivative, apharmaceutically acceptable salt or prodrug thereof, or combinationsthereof to a patient in need thereof.

In certain embodiments, said diseases are selected from the groupconsisting of peripheral denervation syndromes, trauma, amyotrophiclateral sclerosis, meningitis, and structural abnormalities of thespine.

Also provided herein is a method of treating diseases associated withloss of number, loss of function, or loss of correct, optimallyefficient internal organization of skeletal muscle cells or cardiacmuscle cells, comprising the administration of a therapeuticallyeffective amount of epicatechin, either (+) or (−) enantiomers, or acombination of both, an epicatechin derivative, a pharmaceuticallyacceptable salt or prodrug thereof, or combinations thereof to a patientin need thereof. Also provided is a composition for treating diseasesassociated with loss of number, loss of function, or loss of correct,optimally efficient internal organization of skeletal muscle cells orcardiac muscle cells, comprising a therapeutically effective amount ofepicatechin, either (+) or (−) enantiomers, or a combination of both, anepicatechin derivative, a pharmaceutically acceptable salt or prodrugthereof, or combinations thereof to a patient in need thereof.

In certain embodiments, said disease is muscle wasting.

In certain embodiments, said disease is sarcopenia.

In certain embodiments, said sarcopenia is associated with aging,diabetes, abnormal metabolic conditions, infection, inflammation,autoimmune, disease, cardiac dysfunction, arthritis congestive heartfailure, aging, myocarditis, myositis, polymyalgia rheumatic,polymyositis, HIV, cancer, side effects of chemotherapy, malnutrition,aging, inborn errors of metabolism, trauma, stroke, and neurologicalimpairment.

In certain embodiments, the method of treating diseases associated withloss of number, loss of function, or loss of correct, optimallyefficient internal organization of skeletal muscle cells or cardiacmuscle cells further comprises exercise or programmatic sequences orintensities of exercise.

Also provided herein is a method of enhancing sports performance,endurance, building muscle shape or strength, or facilitating recoveryfrom the effects of training or competition, comprising theadministration of a therapeutically effective amount of epicatechin,either (+) or (−) enantiomers or a combination of both, an epicatechinderivative, a pharmaceutically acceptable salt or prodrug thereof, orcombinations thereof to a patient in need thereof. Also provided is acomposition for enhancing sports performance, endurance, building muscleshape or strength, or facilitating recovery from the effects of trainingor competition, comprising a therapeutically effective amount ofepicatechin, either (+) or (−) enantiomers or a combination of both, anepicatechin derivative, a pharmaceutically acceptable salt or prodrugthereof, or combinations thereof to a patient in need thereof.

Also provided herein is a method of treating muscle injury, weakness, orpain associated with the administration of medicines, comprising theadministration of a therapeutically effective amount of epicatechin,either (+) or (−) enantiomers or a combination of both, an epicatechinderivative, a pharmaceutically acceptable salt or prodrug thereof, orcombinations thereof to a patient in need thereof. Also provided is acomposition for treating muscle injury, weakness, or pain associatedwith the administration of medicines, comprising a therapeuticallyeffective amount of epicatechin, either (+) or (−) enantiomers or acombination of both, an epicatechin derivative, a pharmaceuticallyacceptable salt or prodrug thereof, or combinations thereof to a patientin need thereof.

In certain embodiments, said medicine is selected from the groupconsisting of corticosteroids such as prednisone, methyl prednisone, orhalogenated derivatives thereof, chemotherapeutics such as doxorubicinor methotrexate, and inhibitors of HMG co-reductase, known as statins,that are frequently associated with muscle disorders or myopathy,including: Advicor′″ (niacin extended-release/lovastatin), Altoprev′″(lovastatin extended-release), Caduet′″ (amlodipine and atorvastatin),Crestor′″ (rosuvastatin), Juvisync′″ (sitagliptin/simvastatin), Lescol®(fluvastatin), Lescol XL (fluvastatin extended-release), Lipitor′″(atorvastatin), Compactin (mevastatin), Livalo® (pitavastatin),Mevacor′″ (lovastatin), Pravachol′″ (pravastatin), Simcor′″ (niacinextended-release/simvastatin), Vytorin′″ (ezetimibe/simvastatin), andZocor® (simvastatin).

In certain embodiments of anyone of the embodiments disclosed above,said epicatechin is substantially (−)-epicatechin.

In certain embodiments of anyone of the embodiments disclosed above,said epicatechin is substantially (+)-epicatechin.

In certain embodiments of anyone of the embodiments disclosed above,said epicatechin is a racemic mixture of (−)-epicatechin and(+)-epicatechin.

In certain embodiments of anyone of the embodiments disclosed above,said patient is selected for treatment based on the occurrence of one ormore physiological manifestations of skeletal or cardiac muscle injuryor dysfunction in the subject.

In further embodiments, said manifestation is elevation in a biomarkerselected from the group consisting of elevated plasma levels ofmyoglobin, troponin, or creatine phosphokinase, lactic acidosis, andcreatinine.

In certain embodiments of anyone of the embodiments disclosed above, adiagnostic biomarker is used to determine the time and degree of muscleresponse.

In further embodiments, said diagnostic biomarker is dystrophin orthromobospondin.

In certain embodiments of anyone of the embodiments disclosed above,epicatechin is administered.

In certain embodiments of anyone of the embodiments disclosed above, anepicatechin derivative is administered.

In further embodiments, said epicatechin, epicatechin derivative,pharmaceutically acceptable salts and prodrugs thereof, or combinationsthereof, are administered orally.

In other embodiments, said epicatechin, epicatechin derivative,pharmaceutically acceptable salts and prodrugs thereof, or combinationsthereof, are administered parenterally.

In other embodiments, said epicatechin, epicatechin derivative,pharmaceutically acceptable salts and prodrugs thereof, or combinationsthereof, are administered as a neutraceutical.

In further embodiments, epicatechin, epicatechin derivatives,pharmaceutically acceptable salts and prodrugs thereof, or combinationsthereof, are administered in combination with an additional therapeuticsagent. Said additional therapeutic agent is selected from the groupconsisting of hormones which stimulate muscle cell growth, y-aminobutyric acid or its derivatives, dietary protein supplements, anabolicsteroids, biological factors known to enhance the growth, strength,endurance, or metabolism of skeletal or cardiac muscle, or recovery ofskeletal muscle or cardiac muscle from injury or weakness, compoundsknown to be associated with increased nitric oxide production whichpromotes blood flow through muscles, extracts of natural products knownto promote muscle strength or endurance, inhibitors of myostatin, andstimulators of follistatin expression.

Also provided herein is a method of diagnosing the degree of severity ofa muscle disorder, comprising the step of measuring the plasma levels offollistatin, myostatin, or the ratio of follistatin to myostatin.

Also provided herein is a method of determining the extent of responseto therapy for a muscle disorder, comprising the steps of:

-   -   a) measuring the pre-treatment plasma levels of follistatin,        myostatin, or the ratio of follistatin to myostatin;    -   b) measuring the post-treatment plasma levels of follistatin,        myostatin, or the ratio of follistatin to myostatin; and    -   c) comparing the pre- and post-treatment levels of follistatin,        myostatin, or the ratio of follistatin to myostatin.

Also provided herein is a method of treatment of a muscle disorder,comprising the steps of:

-   -   a) measuring the plasma levels of follistatin, myostatin, or the        ratio of follistatin to myostatin a first time;    -   b) administering a first amount of epicatechin (either (+) or        (−) enantiomers, or a combination of both), an epicatechin        derivative, or a pharmaceutically acceptable salt or prodrug        thereof;    -   c) measuring the post-treatment plasma levels of follistatin,        myostatin, or the ratio of follistatin to myostatin;    -   d) comparing the pre- and post-treatment levels of follistatin,        myostatin, or the ratio of follistatin to myostatin; and    -   e) either:        -   i) increasing the dose of epicatechin (either (+) or (−)            enantiomers, or a combination of both), an epicatechin            derivative, or a pharmaceutically acceptable salt or prodrug            thereof administered in step b when the measured follistatin            concentration in the subject has increased, when the            measured myostatin concentration in the subject has            decreased, or when the ratio of plasma follistatin to plasma            myostatin has increased; or        -   ii) decreasing or maintaining the dose of epicatechin            (either (+) or (−) enantiomers, or a combination of both),            an epicatechin derivative, or a pharmaceutically acceptable            salt or prodrug thereof administered in step b when the            measured follistatin concentration in the subject has            decreased, when the measured myostatin concentration in the            subject has increased, or when the ratio of plasma            follistatin to plasma myostatin has decreased.

In certain embodiments of anyone of the embodiments disclosed above,said epicatechin is a racemic mixture of greater than 50%(−)-epicatechin and less than 50% (+)-epicatechin.

In certain embodiments of anyone of the embodiments disclosed above,said a racemic mixture is greater than 75% (−)-epicatechin.

In certain embodiments of anyone of the embodiments disclosed above,said a racemic mixture is greater than 90% (−)-epicatechin.

In certain embodiments of anyone of the embodiments disclosed above,said a racemic mixture is greater than 75% (+)-epicatechin.

In certain embodiments of anyone of the embodiments disclosed above,said a racemic mixture greater than 90% (+)-epicatechin.

Also provided herein is the use of epicatechin, (+)-epicatechin,(−)-epicatechin, a combination of (+)- and (−)-epicatechin, anepicatechin derivative, a pharmaceutically acceptable salt or prodrug ofany of the foregoing, or a combination of any of the foregoing, in themanufacture of a medicament for the treatment of any of the diseases, orfor the achievement of any therapeutic or functional endpoint, asdisclosed herein.

As used herein, the terms below have the meanings indicated.

When ranges of values are disclosed, and the notation “from n, . . . ton,” or “between n, . . . and n,” is used, where n, and n2 are thenumbers, then unless otherwise specified, this notation is intended toinclude the numbers themselves and the range between them. This rangemay be integral or continuous between and including the end values. Byway of example, the range “from 2 to 6 carbons” is intended to includetwo, three, four, five, and six carbons, since carbons come in integerunits. Compare, by way of example, the range “from 1 to 3 uM(micromolar),” which is intended to include 1 uM, 3 uM, and everythingin between to any number of significant figures (e.g., 1.255 uM, 2.1 uM,2.9999 uM, etc.).

The term “about,” as used herein, is intended to qualify the numericalvalues which it modifies, denoting such a value as variable within amargin of error. When no particular margin of error, such as a standarddeviation to a mean value given in a chart or table of data, is recited,the term “about” should be understood to mean that range which wouldencompass the recited value and the range which would be included byrounding up or down to that figure as well, taking into accountsignificant figures.

The term “disease” as used herein is intended to be generallysynonymous, and is used interchangeably with, the terms “disorder,”“syndrome,” and “condition” (as in medical condition), in that allreflect an abnormal condition of the human or animal body or of one ofits parts that impairs normal functioning, is typically manifested bydistinguishing signs and symptoms, and causes the human or animal tohave a reduced duration or quality of life.

The term “muscular diseases” refers to diseases associated with impairedskeletal muscle or cardiac muscle cell number or function.

The term “combination therapy” means the administration of two or moretherapeutic agents to treat a therapeutic condition or disorderdescribed in the present disclosure. Such administration encompassesco-administration of these therapeutic agents in a substantiallysimultaneous manner, such as in a single capsule having a fixed ratio ofactive ingredients or in multiple, separate capsules for each activeingredient. In addition, such administration also encompasses use ofeach type of therapeutic agent in a sequential manner. In either case,the treatment regimen will provide beneficial effects of the drugcombination in treating the conditions or disorders described herein. Incertain embodiments, a combination of compounds is administered suchthat the clearance half-life of each compound from the body overlaps atleast partially with one another. For example, a first pharmaceuticalhas a clearance half-life of 1 hour and is administered at time=O, and asecond pharmaceutical has a clearance half-life of 1 hour and isadministered at time=45 minutes.

The phrase “therapeutically effective” is intended to qualify the amountof active ingredients used in the treatment of a disease or disorder oron the effecting of a clinical endpoint.

The term “therapeutically acceptable” refers to those compounds (orsalts, prodrugs, tautomers, zwitterionic forms, etc.) which are suitablefor use in contact with the tissues of patients without undue toxicity,irritation, and allergic response, are commensurate with a reasonablebenefit/risk ratio, and are effective for their intended use.

As used herein, reference to “treatment” of a patient is intended toinclude prophylaxis. Treatment may also be preemptive in nature, i.e.,it may include prevention of disease. Prevention of a disease mayinvolve complete protection from disease, for example as in the case ofprevention of infection with a pathogen, or may involve prevention ofdisease progression. For example, prevention of a disease may not meancomplete foreclosure of any effect related to the diseases at any level,but instead may mean prevention of the symptoms of a disease to aclinically significant or detectable level. Prevention of diseases mayalso mean prevention of progression of a disease to a later stage of thedisease.

The term “patient” is generally synonymous with the term “subject” andincludes all mammals including humans. Examples of patients includehumans, livestock such as cows, goats, sheep, pigs, and rabbits, andcompanion animals such as dogs, cats, rabbits, and horses. Preferably,the patient is a human.

The term “epicatechin” as used herein refers to (+)-epicatechin (2R-3R),(−)-epicatechin (2S-3S), or mixtures thereof. In certain embodiments,“epicatechin” refers to (+)-epicatechin. In further embodiments,“epicatechin” refers to (−)-epicatechin. In further embodiments,“epicatechin” refers to a racemic mixture of (+)-epicatechin and(−)-epicatechin.

The term “epicatechin derivative” as used herein refers to any compoundwhich retains the ring structure and stereochemistry of epicatechinitself, but which contains one or more substituent groups relative toepicatechin. Certain naturally occurring epicatechin derivatives areknown, such as (−)-epigallocatechin (EGC), (−)-epicatechin-3-gallate(ECG), (−)-epigallocatechin-3-gallate (EGCG), (+)-epigallocatechin(EGC), (+)-epicatechin-3-gallate (ECG), and(+)-epigallocatechin-3-gallate (EGCG). The term also includescombination molecules or prodrugs that release epicatechin or aderivative thereof when administered to a subject. Such a combinationmolecule may include, for example, epicatechin and an agent joined by ahydrolysable linker group.

Epicatechin and its derivatives may be made synthetically, or may beisolated from natural sources that contain these compounds, such aschocolate, tea, and nuts. The term “chocolate” as used herein refers toa solid or semi-plastic food and is intended to refer to all chocolateor chocolate-like compositions containing a dispersion of solids withina fat phase. The term is intended to include compositions conforming tothe U.S. Standards of Identity (501), CODEX Alimentarius and/or otherinternational standards and compositions not conforming to the U.S.Standards of Identity or other international standards. The term“chocolate” encompasses sweet chocolate, bittersweet or semisweetchocolate, milk chocolate, buttermilk chocolate, skim milk chocolate,mixed dairy product chocolate, sweet cocoa and vegetable fat coating,sweet chocolate and vegetable fat coating, milk chocolate and vegetablefat coating, vegetable fat based coating, pastels including whitechocolate or coating made with cocoa butter or vegetable fat or acombination of these, nutritionally modified chocolate-like compositions(chocolates or coatings made with reduced calorie ingredients), and lowfat chocolates, unless specifically identified otherwise. See, e.g.,U.S. Pat. No. 6,312,753, which is hereby incorporated by referenceherein. By way of example, epicatechin and its derivatives may bedelivered by administration of tea extracts, cocoa components, partiallyand fully defatted cocoa solids, cocoa nibs and fractions derivedtherefrom, cocoa polyphenol extracts, cocoa butter, chocolate liquors,and mixtures thereof.

The term “prodrug” refers to a compound that is made more active invivo. Certain compounds disclosed herein may also exist as prodrugs, asdescribed in Hydrolysis in Drug and Prodrug Metabolism: Chemistry,Biochemistry, and Enzymology (Testa, Bernard and Mayer, Joachim M.Wiley-VHCA, Zurich, Switzerland 2003). Prodrugs of the compoundsdescribed herein are structurally modified forms of the compound thatreadily undergo chemical changes under physiological conditions toprovide the compound. Additionally, prodrugs can be converted to thecompound by chemical or biochemical methods in an ex vivo environment.For example, prodrugs can be slowly converted to a compound when placedin a transdermal patch reservoir with a suitable enzyme or chemicalreagent. Prodrugs are often useful because, in some situations, they maybe easier to administer than the compound, or parent drug. They may, forinstance, be bioavailable by oral administration whereas the parent drugis not. The prodrug may also have improved solubility in pharmaceuticalcompositions over the parent drug. A wide variety of prodrug derivativesare known in the art, such as those that rely on hydrolytic cleavage oroxidative activation of the prodrug. An example, without limitation, ofa prodrug would be a compound which is administered as an ester (the“prodrug”), but then is metabolically hydrolyzed to the carboxylic acid,the active entity. Additional examples include peptidyl derivatives of acompound.

The compounds disclosed herein can exist as therapeutically acceptablesalts. The present invention includes compounds listed above in the formof salts, including acid addition salts. Suitable salts include thoseformed with both organic and inorganic acids. Such acid addition saltswill normally be pharmaceutically acceptable. However, salts ofnon-pharmaceutically acceptable salts may be of utility in thepreparation and purification of the compound in question. Basic additionsalts may also be formed and be pharmaceutically acceptable. For a morecomplete discussion of the preparation and selection of salts, refer toPharmaceutical Salts: Properties, Selection, and Use (Stahl, P.Heinrich. Wiley-VCHA, Zurich, Switzerland, 2002).

The term “therapeutically acceptable salt,” as used herein, representssalts or zwitterionic forms of the compounds disclosed herein which arewater or oil-soluble or dispersible and therapeutically acceptable asdefined herein. The salts can be prepared during the final isolation andpurification of the compounds or separately by reacting the appropriatecompound in the form of the free base with a suitable acid.Representative acid addition salts include acetate, adipate, alginate,L-ascorbate, aspartate, benzoate, benzenesulfonate (besylate),bisulfate, butyrate, camphorate, camphorsulfonate, citrate, digluconate,formate, fumarate, gentisate, glutarate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isethionate),lactate, maleate, malonate, DL-mandelate, mesitylenesulfonate, methanesulfonate, naphthy lenesulfonate, nicotinate, 2-naphthalenesulfonate,oxalate, pamoate, pectinate, persulfate, 3-phenylproprionate,phosphonate, picrate, pivalate, propionate, pyroglutamate, succinate,sulfonate, tartrate, L-tartrate, trichloroacetate, trifluoroacetate,phosphate, glutamate, bicarbonate, para-toluene sulfonate (p-tosylate),and undecanoate. Also, basic groups in the compounds disclosed hereincan be quaternized with methyl, ethyl, propyl, and butyl chlorides,bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates;decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides;and benzyl and phenethyl bromides. Examples of acids which can beemployed to form therapeutically acceptable addition salts includeinorganic acids such as hydrochloric, hydrobromic, sulfuric, andphosphoric, and organic acids such as oxalic, maleic, succinic, andcitric. Salts can also be formed by coordination of the compounds withan alkali metal or alkaline earth ion. Hence, the present inventioncontemplates sodium, potassium, magnesium, and calcium salts of thecompounds disclosed herein, and the like.

Basic addition salts can be prepared during the final isolation andpurification of the compounds by reacting a carboxy group with asuitable base such as the hydroxide, carbonate, or bicarbonate of ametal cation or with ammonia or an organic primary, secondary, ortertiary amine. The cations of therapeutically acceptable salts includelithium, sodium, potassium, calcium, magnesium, and aluminum, as well asnontoxic quaternary amine cations such as ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, diethylamine, ethylamine, tributylamine, pyridine,N,N-dimethy laniline, N-methy 1piperidine, N-methylmorpholine,dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine,1-ephenamine, and N,N-dibenzylethylenediamine. Other representativeorganic amines useful for the formation of base addition salts includeethylenediamine, ethanolamine, diethanolamine, piperidine, andpiperazine.

A salt of a compound can be made by reacting the appropriate compound inthe form of the free base with the appropriate acid.

While it may be possible for the compounds of the subject invention tobe administered as the raw chemical, it is also possible to present themas a pharmaceutical formulation. Accordingly, provided herein arepharmaceutical formulations which comprise one or more of certaincompounds disclosed herein, or one or more pharmaceutically acceptablesalts, esters, prodrugs, amides, or solvates thereof, together with oneor more pharmaceutically acceptable carriers thereof and optionally oneor more other therapeutic ingredients. The carriers) must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof. Properformulation is dependent upon the route of administration chosen. Any ofthe well-known techniques, carriers, and excipients may be used assuitable and as understood in the art; e.g., in Remington'sPharmaceutical Sciences. The pharmaceutical compositions disclosedherein may be manufactured in any manner known in the art, e.g., bymeans of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or compressionprocesses.

The formulations include those suitable for oral, parenteral (includingsubcutaneous, intradermal, intramuscular, intravenous, intraarticular,and intramedullary), intraperitoneal, transmucosal, transdermal, rectaland topical (including dermal, buccal, sublingual and intraocular)administration although the most suitable route may depend upon forexample the condition and disorder of the recipient. The formulationsmay conveniently be presented in unit dosage form and may be prepared byany of the methods well known in the art of pharmacy. Typically, thesemethods include the step of bringing into association a compound of thesubject invention or a pharmaceutically acceptable salt, ester, amide,prodrug or solvate thereof (“active ingredient”) with the carrier whichconstitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both and then, if necessary, shaping the product intothe desired formulation.

Formulations of the compounds disclosed herein suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or a suspension in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water-in-oil liquid emulsion. The active ingredient mayalso be presented as a bolus, electuary or paste.

Pharmaceutical preparations which can be used orally include tablets,push-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. Tablets maybe made by compression or molding, optionally with one or more accessoryingredients. Compressed tablets may be prepared by compressing in asuitable machine the active ingredient in a free-flowing form such as apowder or granules, optionally mixed with binders, inert diluents, orlubricating, surface active or dispersing agents. Molded tablets may bemade by molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets may optionally becoated or scored and may be formulated so as to provide slow orcontrolled release of the active ingredient therein. All formulationsfor oral administration should be in dosages suitable for suchadministration. The push-fit capsules can contain the active ingredientsin admixture with filler such as lactose, binders such as starches,and/or lubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds may be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycols. In addition, stabilizers may be added.Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Specific sustained release formulations of the compounds disclosedherein are described in U.S. Pat. No. 6,410,052, which is herebyincorporated by reference.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. The formulations may be presentedin unit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in powder form or in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example, saline or sterile pyrogen-free water,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Formulations for parenteral administration include aqueous andnon-aqueous (oily) sterile injection solutions of the active compoundswhich may contain antioxidants, buffers, bacteriostats and solutes whichrender the formulation isotonic with the blood of the intendedrecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and thickening agents. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

For buccal or sublingual administration, the compositions may take theform of tablets, lozenges, pastilles, or gels formulated in conventionalmanner. Such compositions may comprise the active ingredient in aflavored basis such as sucrose and acacia or tragacanth.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter, polyethylene glycol, or otherglycerides.

Certain compounds disclosed herein may be administered topically, thatis by non-systemic administration. This includes the application of acompound disclosed herein externally to the epidermis or the buccalcavity and the instillation of such a compound into the ear, eye andnose, such that the compound does not significantly enter the bloodstream. In contrast, systemic administration refers to oral,intravenous, intraperitoneal and intramuscular administration.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site of inflammation such as gels, liniments, lotions, creams,ointments or pastes, and drops suitable for administration to the eye,ear or nose. The active ingredient for topical administration maycomprise, for example, from 0.001% to 10% w/w (by weight) of theformulation. In certain embodiments, the active ingredient may compriseas much as 10% w/w. In other embodiments, it may comprise less than 5%w/w. In certain embodiments, the active ingredient may comprise from 2%w/w to 5% w/w. In other embodiments, it may comprise from 0.1% to 1% w/wof the formulation.

For administration by inhalation, compounds may be convenientlydelivered from an insufflator, nebulizer pressurized packs or otherconvenient means of delivering an aerosol spray. Pressurized packs maycomprise a suitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Alternatively, for administration by inhalation or insufflation, thecompounds according to the invention may take the form of a dry powdercomposition, for example a powder mix of the compound and a suitablepowder base such as lactose or starch. The powder composition may bepresented in unit dosage form, in for example, capsules, cartridges,gelatin or blister packs from which the powder may be administered withthe aid of an inhalator or insufflator.

Preferred unit dosage formulations are those containing an effectivedose, as herein below recited, or an appropriate fraction thereof, ofthe active ingredient.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations described above may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavoring agents.

Compounds may be administered orally or via injection at a dose of from0.1 to 500 mg/kg per day. The dose range for adult humans is generallyfrom 5 mg to 2 g/day. Tablets or other forms of presentation provided indiscrete units may conveniently contain an amount of one or morecompounds which is effective at such dosage or as a multiple of thesame, for instance, units containing 5 mg to 500 mg, usually around 10mg to 200 mg.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration.

The compounds can be administered in various modes, e.g. orally,topically, or by injection. The precise amount of compound administeredto a patient will be the responsibility of the attendant physician. Thespecific dose level for any particular patient will depend upon avariety of factors including the activity of the specific compoundemployed, the age, body weight, general health, sex, diets, time ofadministration, route of administration, rate of excretion, drugcombination, the precise disorder being treated, and the severity of theindication or condition being treated. Also, the route of administrationmay vary depending on the condition and its severity.

In certain instances, it may be appropriate to administer at least oneof the compounds described herein (or a pharmaceutically acceptablesalt, ester, or prodrug thereof) in combination with another therapeuticagent. By way of example only, if one of the side effects experienced bya patient upon receiving one of the compounds herein is hypertension,then it may be appropriate to administer an anti-hypertensive agent incombination with the initial therapeutic agent. Or, by way of exampleonly, the therapeutic effectiveness of one of the compounds describedherein may be enhanced by administration of an adjuvant (i.e., by itselfthe adjuvant may only have minimal therapeutic benefit, but incombination with another therapeutic agent, the overall therapeuticbenefit to the patient is enhanced). Or, by way of example only, thebenefit of experienced by a patient may be increased by administeringone of the compounds described herein with another therapeutic agent(which also includes a therapeutic regimen) that also has therapeuticbenefit. By way of example only, in a treatment for diabetes involvingadministration of one of the compounds described herein, increasedtherapeutic benefit may result by also providing the patient withanother therapeutic agent for diabetes. In any case, regardless of thedisease, disorder or condition being treated, the overall benefitexperienced by the patient may simply be additive of the two therapeuticagents or the patient may experience a synergistic benefit.

Specific, non-limiting examples of possible combination therapiesinclude use of certain compounds of the invention with agents whichallow or enhance improvements in the number, structure or function ofskeletal muscle cells or cardiac muscle cells.

In further embodiments, such agents include hormones which stimulatemuscle cell growth, butyric acid or its derivatives, dietary proteinsupplements, anabolic steroids, biological factors known to enhance thegrowth, strength, endurance, or metabolism of skeletal or cardiacmuscle, or recovery of skeletal muscle or cardiac muscle from injury orweakness, compounds known to be associated with increased nitric oxideproduction which promotes blood flow through muscles, extracts ofnatural products known to promote muscle strength or endurance,inhibitors of myostatin, and stimulators of folistatin expression.

In further embodiments, hormones which stimulate muscle cell growthinclude, but are not limited to, growth hormone, growth hormone analogs,growth hormone releasing peptides or analogs thereof, growth hormonesecretagogues, or other hormones such as somatatropin or mechano growthfactor.

In further embodiments butyric acid derivatives includeneurotransmitters that benefit muscles by modulating the pituitarygland.

In further embodiments, dietary protein supplements include, but are notlimited to, proteins such as casein, amino acids precursors orderivatives thereof with known attributes of potentiating muscle growth,such as leucine, valine, isovaline, beta alanine, glutamine, glutaminedipeptide, or glycocyamine.

In further embodiments anabolic steroids, include, but are not limitedto, testosterone or related steroid compounds with muscle growthinducing properties, such as cyclostanazol or methadrostenol, prohomonesor derivatives thereof, modulators of estrogen, and selective androgenreceptor modulators (SARMS).

In further embodiments, biological factors known to enhance the growth,strength, endurance, or metabolism of skeletal or cardiac muscle, orrecovery of skeletal muscle or cardiac muscle from injury or weakness,include, but are not limited to, alpha-lipoic acid, taurine, caffeine,magnesium, niacin, folic acid, ornithine, vitamin B6, B12, or D,aspartate, creatine and its diverse salts such creatine monohydrate,betaine, N-acetyl cysteine, beta-hydroxyl methyl butyrate, lecithin,choline, phospholipid mixtures, phosphatidyl serine, carnitine,L-carnitine, and glycine proprionyl-L-carnitine.

In further embodiments, compounds known to be associated with increasednitric oxide production which promotes blood flow through musclesinclude, but are not limited to, arginine and citrulline.

In further embodiments, extracts of natural products known to promotemuscle strength or endurance, include, but are not limited to, guarana,geranium Robertianum, Cirsium ologophyllum, Bauhinia purpureae, Yohimbe,Bacopa monniera, beet powder, rhodiola, or tea extracts.

In further embodiments, inhibitors of myostatin are proteins,antibodies, peptides, or small molecules.

In further embodiments, stimulators of folistatin expression or functionare proteins, peptides, or small molecules.

In any case, the multiple therapeutic agents (at least one of which is acompound disclosed herein) may be administered in any order or evensimultaneously. If simultaneously, the multiple therapeutic agents maybe provided in a single, unified form, or in multiple forms (by way ofexample only, either as a single pill or as two separate pills). One ofthe therapeutic agents may be given in multiple doses, or both may begiven as multiple doses. If not simultaneous, the timing between themultiple doses may be any duration of time ranging from a few minutes tofour weeks.

Thus, in another aspect, certain embodiments provide methods fortreating muscular diseases in a human or animal subject in need of suchtreatment comprising administering to said subject an amount of acompound disclosed herein effective to reduce or prevent said disorderin the subject, in combination with at least one additional agent forthe treatment of said disorder that is known in the art. In a relatedaspect, certain embodiments provide therapeutic compositions comprisingat least one compound disclosed herein in combination with one or moreadditional agents for the treatment of muscular diseases.

The compositions of the present invention may also be formulated asneutraceutical compositions. The term “neutraceutical composition” asused herein refers to a food product, foodstuff, dietary supplement,nutritional supplement or a supplement composition for a food product ora foodstuff comprising exogenously added catechin and/or epicatechin.Details on techniques for formulation and administration of suchcompositions may be found in Remington, The Science and Practice ofPharmacy 21st Edition (Mack Publishing Co., Easton, Pa.) and Nielloudand Marti-Mestres, Pharmaceutical Emulsions and Suspensions: 2nd Edition(Marcel Dekker, Inc, New York).

As used herein, the term food product refers to any food or feedsuitable for consumption by humans or animals. The food product may be aprepared and packaged food (e.g., mayonnaise, salad dressing, bread,grain bar, beverage, etc.) or an animal feed (e.g., extruded andpelleted animal feed, coarse mixed feed or pet food composition). Asused herein, the term foodstuff refers to any substance fit for human oranimal consumption.

Food products or foodstuffs are for example beverages such asnonalcoholic and alcoholic drinks as well as liquid preparation to beadded to drinking water and liquid food, non-alcoholic drinks are forinstance soft drinks, sport drinks, fruit juices, such as for exampleorange juice, apple juice and grapefruit juice; lemonades, teas,near-water drinks and milk and other dairy drinks such as for exampleyoghurt drinks, and diet drinks. In another embodiment food products orfoodstuffs refer to solid or semi-solid foods comprising the compositionaccording to the invention. These forms can include, but are not limitedto baked goods such as cakes and cookies, puddings, dairy products,confections, snack foods, or frozen confections or novelties (e.g., icecream, milk shakes), prepared frozen meals, candy, snack products (e.g.,chips), liquid food such as soups, spreads, sauces, salad dressings,prepared meat products, cheese, yogurt and any other fat or oilcontaining foods, and food ingredients (e.g., wheat flour).

Animal feed including pet food compositions advantageously include foodintended to supply necessary dietary requirements, as well as treats(e.g., dog biscuits) or other food supplements. The animal feedcomprising the composition according to the invention may be in the formof a dry composition (for example, kibble), semi-moist composition, wetcomposition, or any mixture thereof. Alternatively or additionally, theanimal feed is a supplement, such as a gravy, drinking water, yogurt,powder, suspension, chew, treat (e.g., biscuits) or any other deliveryform.

The term “dietary supplement” refers to a small amount of a compound forsupplementation of a human or animal diet packaged in single or multipledose units.

Dietary supplements do not generally provide significant amounts ofcalories but may contain other micronutrients (e.g., vitamins orminerals). The term food products or foodstuffs also includes functionalfoods and prepared food products pre-packaged for human consumption.

The term nutritional supplement refers to a composition comprising adietary supplement in combination with a source of calories. In someembodiments, nutritional supplements are meal replacements orsupplements (e.g., nutrient or energy bars or nutrient beverages orconcentrates).

Dietary supplements of the present invention may be delivered in anysuitable format. In certain embodiments, dietary supplements areformulated for oral delivery. The ingredients of the dietary supplementof this invention are contained in acceptable excipients and/or carriersfor oral consumption. The actual form of the carrier, and thus, thedietary supplement itself, is not critical. The carrier may be a liquid,gel, gelcap, capsule, powder, solid tablet (coated or noncoated), tea,or the like.

In certain embodiments, the dietary supplement is in the form of atablet or capsule and in further embodiments is in the form of a hard(shell) capsule. Suitable excipient and/or carriers includemaltodextrin, calcium carbonate, dicalcium phosphate, tricalciumphosphate, microcrystalline cellulose, dextrose, rice flour, magnesiumstearate, stearic acid, croscarmellose sodium, sodium starch glycolate,crospovidone, sucrose, vegetable gums, lactose, methylcellulose,povidone, carboxymethylcellulose, corn starch, and the like (includingmixtures thereof). In certain embodiments, carriers include calciumcarbonate, magnesium stearate, maltodextrin, and mixtures thereof. Thevarious ingredients and the excipient and/or carrier are mixed andformed into the desired form using conventional techniques. The tabletor capsule of the present invention may be coated with an entericcoating that dissolves at a pH of about 6.0 to 7.0. A suitable entericcoating that dissolves in the small intestine but not in the stomach iscellulose acetate phthalate.

In other embodiments, the dietary supplement is provided as a powder orliquid suitable for adding by the consumer to a food or beverage. Forexample, in some embodiments, the dietary supplement can be administeredto an individual in the form of a powder, for instance to be used bymixing into a beverage, or by stirring into a semi-solid food such as apudding, topping, sauce, puree, cooked cereal, or salad dressing, forinstance, or by otherwise adding to a food or the dietary supplemente.g. enclosed in caps of food or beverage container for releaseimmediately before consumption. The dietary supplement may comprise oneor more inert ingredients, especially if it is desirable to limit thenumber of calories added to the diet by the dietary supplement. Forexample, the dietary supplement of the present invention may alsocontain optional ingredients including, for example, herbs, vitamins,minerals, enhancers, colorants, sweeteners, flavorants, inertingredients, and the like.

In some embodiments, the dietary supplements further comprise vitaminsand minerals including, but not limited to, calcium phosphate oracetate, tribasic; potassium phosphate, dibasic; magnesium sulfate oroxide; salt (sodium chloride); potassium chloride or acetate; ascorbicacid; ferric orthophosphate; niacinamide; zinc sulfate or oxide; calciumpantothenate; copper gluconate; riboflavin; beta-carotene; pyridoxinehydrochloride; thiamin mononitrate; folic acid; biotin; chromiumchloride or picolonate; potassium iodide; sodium selenate; sodiummolybdate; phylloquinone; vitamin D3; cyanocobalamin; sodium selenite;copper sulfate; vitamin A; vitamin C; inositol; potassium iodide.Suitable dosages for vitamins and minerals may be obtained, for example,by consulting the U.S. RDA guidelines.

In other embodiments, the present invention provides nutritionalsupplements (e.g., energy bars or meal replacement bars or beverages)comprising the composition according to the invention. The nutritionalsupplement may serve as meal or snack replacement and generally providenutrient calories. In certain embodiments, the nutritional supplementsprovide carbohydrates, proteins, and fats in balanced amounts. Thenutritional supplement can further comprise carbohydrate, simple, mediumchain length, or polysaccharides, or a combination thereof. A simplesugar can be chosen for desirable organoleptic properties. Uncookedcornstarch is one example of a complex carbohydrate. If it is desiredthat it should maintain its high molecular weight structure, it shouldbe included only in food formulations or portions thereof which are notcooked or heat processed since the heat will break down the complexcarbohydrate into simple carbohydrates, wherein simple carbohydrates aremono or disaccharides. The nutritional supplement contains, in oneembodiment, combinations of sources of carbohydrate of three levels ofchain length (simple, medium and complex; e.g., sucrose, maltodextrins,and uncooked cornstarch).

Sources of protein to be incorporated into the nutritional supplement ofthe invention can be any suitable protein utilized in nutritionalformulations and can include whey protein, whey protein concentrate,whey powder, egg, soy flour, soy milk soy protein, soy protein isolate,caseinate (e.g., sodium caseinate, sodium calcium caseinate, calciumcaseinate, potassium caseinate), animal and vegetable protein andhydrolysates or mixtures thereof. When choosing a protein source, thebiological value of the protein should be considered first, with thehighest biological values being found in caseinate, whey, lactalbumin,egg albumin and whole egg proteins. In an embodiment, the protein is acombination of whey protein concentrate and calcium caseinate. Theseproteins have high biological value; that is, they have a highproportion of the essential amino acids. See Modern Nutrition in Healthand Disease, 8th ed., Lea & Febiger, 1986, especially Volume 1, pages30-32.

The nutritional supplement can also contain other ingredients, such asone or a combination of other vitamins, minerals, antioxidants, fiberand other dietary supplements (e.g., protein, amino acids, choline,lecithin). Selection of one or several of these ingredients is a matterof formulation, design, consumer preferences and end user. The amountsof these ingredients added to the dietary supplements of this inventionare readily known to the skilled artisan. Guidance to such amounts canbe provided by the U.S. RDA doses for children and adults. Furthervitamins and minerals that can be added include, but are not limited to,calcium phosphate or acetate, tribasic; potassium phosphate, dibasic;magnesium sulfate or oxide; salt (sodium chloride); potassium chlorideor acetate; ascorbic acid; ferric orthophosphate; niacinamide; zincsulfate or oxide; calcium pantothenate; copper gluconate; riboflavin;beta-carotene; pyridoxine hydrochloride; thiamin mononitrate; folicacid; biotin; chromium chloride or picolonate; potassium iodide; sodiumselenate; sodium molybdate; phylloquinone; vitamin D3; cyanocobalamin;sodium selenite; copper sulfate; vitamin A; vitamin C; inositol;potassium iodide.

The nutritional supplement can be provided in a variety of forms, and bya variety of production methods. In an embodiment, to manufacture a foodbar, the liquid ingredients are cooked; the dry ingredients are addedwith the liquid ingredients in a mixer and mixed until the dough phaseis reached; the dough is put into an extruder, and extruded; theextruded dough is cut into appropriate lengths; and the product iscooled. The bars may contain other nutrients and fillers to enhancetaste, in addition to the ingredients specifically listed herein.

It is understood by those of skill in the art that other ingredients canbe added to those described herein, for example, fillers, emulsifiers,preservatives, etc. for the processing or manufacture of a nutritionalsupplement.

Additionally, flavors, coloring agents, spices, nuts and the like may beincorporated into the neutraceutical composition. Flavorings can be inthe form of flavored extracts, volatile oils, chocolate flavorings,peanut butter flavoring, cookie crumbs, crisp rice, vanilla or anycommercially available flavoring. Examples of useful flavoring include,but are not limited to, pure anise extract, imitation banana extract,imitation cherry extract, chocolate extract, pure lemon extract, pureorange extract, pure peppermint extract, imitation pineapple extract,imitation rum extract, imitation strawberry extract, or pure vanillaextract; or volatile oils, such as balm oil, bay oil, bergamot oil,cedarwood oil, walnut oil, cherry oil, cinnamon oil, clove oil, orpeppermint oil; peanut butter, chocolate flavoring, vanilla cookiecrumb, butterscotch or toffee. In one embodiment, the dietary supplementcontains cocoa or chocolate.

Emulsifiers may be added for stability of the neutraceuticalcompositions. Examples of suitable emulsifiers include, but are notlimited to, lecithin (e.g., from egg or soy), and/or mono anddiglycerides. Other emulsifiers are readily apparent to the skilledartisan and selection of suitable emulsifier(s) will depend, in part,upon the formulation and final product. Preservatives may also be addedto the nutritional supplement to extend product shelf life. In certainembodiments, preservatives such as potassium sorbate, sodium sorbate,potassium benzoate, sodium benzoate or calcium disodium EDTA are used.

In addition to the carbohydrates described above, the neutraceuticalcomposition can contain natural or artificial (preferably low calorie)sweeteners, e.g., saccharides, cyclamates, aspartamine, aspartame,acesulfame K, and/or sorbitol. Such artificial sweeteners can bedesirable if the nutritional supplement is intended to be consumed by anoverweight or obese individual, or an individual with type II diabeteswho is prone to hyperglycemia.

Moreover, a multi-vitamin and mineral supplement may be added to theneutraceutical compositions of the present invention to obtain anadequate amount of an essential nutrient, which is missing in somediets. The multi-vitamin and mineral supplement may also be useful fordisease prevention and protection against nutritional losses anddeficiencies due to lifestyle patterns.

The dosage and ratios of catechin and/or epicatechin and additionalcomponents administered via a neutraceutical will vary depending uponknown factors, such as the pharmaceutical characteristics of theparticular composition and its mode and route of administration; theage, health and weight of the recipient; the nature and extent of thesymptoms; the kind of concurrent treatment; the frequency of treatment;and the effect desired which can determined by the expert in the fieldwith normal trials, or with the usual considerations regarding theformulation of a neutraceutical composition.

It will be understood, however, that the specific dose level for anyparticular patient will depend on a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex and diet of the individual being treated; the timeand route of administration; the rate of excretion; other drugs whichhave previously been administered; and the severity of the particulardisease undergoing therapy, as is well understood by those skilled inthe art.

Specific diseases to be treated by the compounds, compositions, andmethods disclosed herein include: impaired skeletal and cardiac musclefunction, recovery of skeletal or cardiac muscle health or function,functionally significant regeneration of skeletal or cardiac musclecells or function, and any other diseases disclosed herein.

Besides being useful for human treatment, certain compounds andformulations disclosed herein may also be useful for veterinarytreatment of companion animals, exotic animals and farm animals,including mammals, rodents, and the like. More preferred animals includehorses, dogs, and cats.

Biological Activity Assays

Western Blot Assays:

Cells or skeletal muscle tissue samples were homogenized in 50 ul lysisbuffer (1% triton X-100, 20 mmol/L Tris, 140 mmol/L NaCl, 2 mmol/L EDTA,and 0.1% SDS) with protease and phosphatase inhibitor cocktailssupplemented with 1 mmol/L PMSF, 2 mmol/L Na3V04 and 1 mmol/L NaP.Homogenates were passed through an insulin syringe five times, sonicatedfor 30 min at 4DC and centrifuged (12,000×g) for 10 min. The totalprotein content was measured in the supernatant. A total of 40)1 g ofprotein was loaded onto a 5% or 10% SDS-PAGE, electrotransferred,incubated for 1 h in blocking solution (5% nonfat dry milk in TBS plus0.1% Tween 20 [TBS-T]), followed by either a 3-h incubation at roomtemperature or overnight incubation at 4DC with primary antibodies.Primary antibodies were typically diluted 1:1000 or 1:2000 in TBS-T plus5% bovine serum albumin. Membranes were washed (3× for 5 min) in TBS-Tand incubated 1 hat room temperature in the presence of HRP-conjugatedsecondary antibodies diluted 1:10,000 in blocking solution. Membraneswere again washed 3 times in TBS-T, and the immunoblots were developedusing an enhanced chemiluminescence detection kit. The band intensitieswere digitally quantified. All primary antibodies are commerciallyavailable.

Mouse Myoblast Assay

Epicatechin induces follistatin expression, suppresses myostatinexpression and accelerates the differentiation of cultured mousemyoblasts into myotubes. These phenomena are associated with increasedexpression of biomarkers of muscle differentiation, such as myogenin andmyoD. The mouse myoblast cell line, C2C12, was grown to semi-confluencein 6-well tissue culture plates and then exposed to epicatechin (100 nM)for three days using literature-standardized differentiation inducingmedia: DMEM supplemented with 2% horse serum. Proteins were extracted,separated by conventional gel electrophoresis and stained as Westernblots by reacting with commercially available antibodies specific formarkers of muscle cell growth and differentiation. In experimentsexamining the comparative effects of (−) and (+) epicatechin enantiomerson muscle cells, the cells were grown to semi-confluence in 6 wellplates, placed into literature-standardized differentiation mediumcontaining horse serum, and then stimulated for 24 hours with (−) or (+)epicatechin enantiomers, at concentrations ranging from 10 nM to 1000nM. The cells were then harvested as above, and Western blots wereprepared to determine relative expression of PGCla and follistatin,using commercially available, specific, primary antibodies.

Treatment of Diabetic Patients with Epicatechin-Rich Cocoa Products

Five patients with type 2 diabetes and heart failure were providedepicatechin-rich cocoa products (providing—100 mg epicatechiniday) everyday for three months.

Epicatechin rich cocoa increased protein levels of the stimulator ofskeletal muscle growth, follistatin, markers of muscle differentiation,(myogenin, myoD) and suppressed the expression of the inhibitoryprotein, myostatin in biopsy samples of human skeletal muscle afterthree months of treatment. Patients underwent biopsies of theirquadriceps muscle before and after completing treatment. The biopsymaterial was analyzed by Western blots for the analysis of muscleprotein content.

Electron micrographs of quadriceps muscle biopsy samples obtained beforeand after three months of treatment show a severe disruption anddistortion of sarcomere ultrastructure in human skeletal muscle ofdiabetic patients with heart failure, consistent with sarcopenia.Treatment significantly restored sarcomere organization of skeletalmuscle to near normal. This phenomenon was evidenced in all fivepatients as average histology score improved significantly.

Treatment with epicatechin induced increased expression of the activatedform (de-acetylated) of PGC 1a, a transcriptional regulator of musclerepair and regeneration, as well as mitochondrial biogenesis, in thepatients' quadriceps muscles. Treatment also increased the expression ofelements of the sarcoglycan protein family, including dystrophin,dysferlin, and utrophin, consistent with the observed improvement insarcomere morphology. Treatment also increased mitochondrial biogenesis,as evidenced by increased Electron Transport Complex proteins per mg ofquadriceps tissue.

Baseline skeletal muscle thiollevels (by using a Cayman Inc. gluthationeassay kit) in diabetic patients with heart failure manifested a markeddecrease evidencing significant tissue oxidative stress as compared tonormal muscle. Treatment with epicatechin rich cocoa restored totalthiollevels, an indication of a normalization of tissue oxidative stresslevels. Epicatechin treatment also increased quadriceps expression ofsuperoxide dismutase and catalase, important enzymes that protectagainst oxidative muscle injury.

In-vivo Studies in Wild Mice and a Mouse Model of Muscular Dystrophy

Wild type (i.e. normal) and delta sarcoglycan (8-SG) null mice whichdevelop muscular dystrophy were treated by oral gavage for 30 days withepicatechin at 1 mg/kg twice a day, obtained from Sigma-Aldrich, or withwater only (Control). Quadriceps muscle protein samples were analyzed byWestern blots to assess for changes in protein levels of themitochondrial proteins porin, mitofilin, complex V (CV), superoxidedismutase 2 (SOD2), and catalase. In wild type mice, epicatechintreatment increases and in 8-SG null mice prevents the loss ofmitochondrial proteins. In the muscular dystrophy mice, epicatechinincreased the protein expression of both catalase and superoxidedismutase 2 in the heart and quadriceps muscle, important enzymes thatcounter the damaging effects of oxidation injury by decreasing theseverity of oxidation injury.

Delta sarcoglycan (8-SG) muscular dystrophy mice exhibit reducedglutathione content (GSH, by using a Cayman Inc. gluthatione assay kit)in quadriceps muscle, evidencing enhanced tissue oxidative stress.Epicatechin treatment markedly increased muscle GSH levels in both wildtype mice and delta sarcoglycan knock-out mice.

Delta sarcoglycan knock-out muscular dystrophy mice demonstrated amarked increase in PGC 1a, a transcriptional factor that regulatesmuscle repair and regeneration, and regulates mitochondrial biogenesis,in skeletal muscle after treatment with oral epicatechin, 1 mg/kg twicea day for 4 weeks.

In one experiment, elderly wild type mice (26 months) with the muscleimpairment of the elderly were treated for 2 weeks with epicatechin, 1mg/kg twice a day for two weeks. They also demonstrated a significantincrease in PGC1u in the skeletal muscle, with a correlative increase inmitochondrial protein expression.

In one experiment, mdx mice, characterized by the same dystrophinmutation as is seen with Duchenne's muscular dystrophy, were treatedorally with epicatechin, 1 mg/kg twice a day for 4 weeks. Theydemonstrated an increase in muscle strength compared to controls treatedwith water, as determined by a standard timed hanging upside down test.

In the mouse myocyte cell line, C2C12, both (−) and (+) epicatechinenantiomers stimulated the expression of PGC1u and follistatin within 24hr, consistent with activation of transcriptional pathways regulatingmuscle regeneration and expression of the muscle trophic hormone,follistatin.

Biomarkers

The induction of follistatin, a muscle growth hormone and suppression ofmyostatin, an inhibitor of muscle growth, by epicatechin in vitro and invivo suggest that these proteins might be useful biomarkers inmonitoring the effects of epicatechin in vivo. In diabetic patients withheart failure, the ratio of follistatin to myostatin was measured andcalculated before and after treatment with epicatechin rich cocoa. Therewas a statistically significant increase in the folistatin/myostatinratio associated with treatment, indicating an increase in follistatinand a decrease in its natural antagonist, myostatin.

Effect on Body Growth

SD rats of the body weight 50-60 g and age 3-4 weeks were divided into 4groups. Group A animals were part of vehicle control group, Group Banimals were administered Dexamethasone daily; Group C animals weredosed Epicatechin 3 mg/kg followed by dosing of Dexamethasone; Group Danimals were dosed 10 mg/kg Epicatechin followed by Dexamethasone. Alldosing were by subcutaneous (SC) mode of administration. Dosing ofanimals was continued for 36 days. Body weight was measured everyalternate day, overall length was measured weekly. Animals werephotographed periodically to further support data. Feed intake, generalhealth and movement were assessed routinely. On 37^(th) day animals weresacrificed and femur and tibia length were measured using VernierCalipers. Blood was collected and stored.

It has been confirmed that follistatin can have a beneficialameliorating effect on the decrease in body growth associated with theinhibition of bone formation secondary to the toxicity ofcorticosteroids, as shown in Table 1 below.

Tables 1-3: Data on growth of 4-week rates on dexamethasone with orwithout additional concurrent treatment with epicatechin.

Dex = 1.5 mpk Dex = 1 mpk Dex = 0.5 mpk WEIGHT (g) Day 1 Day 11 Day 23Day 35 Rat. No Group Ind Avg Ind Avg Ind Avg Ind Avg 1 CONTROL 58.2 61.2123.9 132.3 211.2 220.7 302.8 318.4 2 64.2 140.7 230.2 333.9 3 DEX 55.459.3 91.1 89.1 88.0 85.7 103.9 96.1 4 57.7 84.3 80.0 93.1 5 64.8 91.989.0 91.5 6 DEX + EPI(3) 59.9 59.2 85.6 84.8 104.0 103.5 185.1 183.3 755.4 81.2 105.2 181.7 8 62.2 87.6 101.3 183.1 9 DEX + EPI(10) 54.6 60.376.9 86.1 93.3 103.8 189.4 199.3 10 65.9 95.3 114.3 209.1

Dex = 1.5 mpk Dex = 1 mpk Dex = 0.5 mpk LENGTH (cm) Day 1 Day 13 Day 23Day 35 Rat. No Group Ind Avg Ind Avg Ind Avg Ind Avg 1 CONTROL #DIV/0!17.4 17.1 18.0 18.3 21.5 20.8 2 16.7 18.5 20 3 DEX #DIV/0! 15.4 15.314.5 14.2 14.5 14.2 4 15.1 14.0 14 5 15.3 14.0 14 6 DEX + EPI(3) #DIV/0!15.2 15.1 15.0 14.8 17.5 17.7 7 15.1 15.0 17.5 8 15.1 14.5 18 9 DEX +EPI(10) #DIV/0! 14.5 14.9 15.5 15.5 18.5 18.8 10 15.2 15.5 19

Bone Length (mm) Femur Length Tibia Length Groups (mm) (mm) Group 1 -Vehicle Control 32.30 40.89 Group 2 - Dexas/c 22.68 28.67 Group 3 - Epi1 - 3 MPK s/c 29.57 34.91 Group 4 - Epi 1 - 10 MPK s/c 31.30 35.37

1. A method to induce increased cellular or muscular or bodilyproduction of follistatin and follistatin-like proteins in order toreverse or ameliorate injury to, or weakness of, or loss of, bone, or toprevent fractures, in a subject in need thereof, comprisingadministering to a subject a therapeutically effective dose of(+)-epicatechin and/or (−)-epicatechin or an epicatechin derivative ofeither (+)-epicatechin or (−)-epicatechin 2-17. (canceled)